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In this study, we addressed the effects of N limitation in Bradyrhizobium japonicum for its association with soybean roots. The wild-type strain LP 3001 grew for six generations with a growth rate of 1.2 day(-1) in a minimal medium with 28 mM mannitol as the carbon source and with the N source [(NH(4))(2)SO(4)] limited to only 20 microM. Under these conditions, the glutamine synthetase (GS) activity was five to six times higher than in similar cultures grown with 1 or 0.1 mM (NH(4))(2)SO(4). The NtrBC-inducible GSII form of this enzyme accounted for 60% of the specific activity in N-starved rhizobia, being negligible in the other two cultures. The exopolysaccharide (EPS) and capsular polysaccharide (CPS) contents relative to cell protein were significantly higher in the N-starved cultures, but on the other hand, the poly-3-hydroxybutyrate level did not rise in comparison with N-sufficient cultures. In agreement with the accumulation of CPS in N-starved cultures, soybean lectin (SBL) binding as well as stimulation of rhizobial adsorption to soybean roots by SBL pretreatment were higher. The last effect was evident only in cultures that had not entered stationary phase. We also studied nodC gene induction in relation to N starvation. In the chromosomal nodC::lacZ fusion Bj110-573, nodC gene expression was induced by genistein 2.7-fold more in N-starved young cultures than in nonstarved ones. In stationary-phase cultures, nodC gene expression was similarly induced in N-limited cultures, but induction was negligible in cultures limited by another nutrient. Nodulation profiles obtained with strain LP 3001 grown under N starvation indicated that these cultures nodulated faster. In addition, as culture age increased, the nodulation efficiency decreased for two reasons: fewer nodules were formed, and nodulation was delayed. However, their relative importance was different according to the nutrient condition: in older cultures the overall decrease in the number of nodules was the main effect in N-starved cultures, whereas a delay in nodulation was more responsible for a loss in efficiency of N-sufficient cultures. Competition for nodulation was studied with young cultures of two wild-type strains differing only in their antibiotic resistance, the N-starved cultures being the most competitive.

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Soybean seed lectin stimulates adsorption of Bradyrhizobium japonicum to its host roots. Pretreatment of the rhizobia with soybean seed lectin for at least 6-12 h previous to their interaction with the plants was required to detect the stimulatory effect. This activity could be observed with as few as 1000 soybean seed lectin molecules per bacterium, and required specific carbohydrate binding. Infectivity and competitiveness for nodulation were also stimulated by preincubation of the rhizobia either with soybean seed meal extract or soybean seed lectin, the extract being more effective in enhancing competitiveness.

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In this work, we evaluated how the use of alternative N sources affects drought-stress tolerance in common beans. To this end, plants were cultivated employing either N(2) fixation or two levels of inorganic nitrogen: 1 mM NH(4)NO(3) (limiting) or 10 mM NH(4)NO(3) (sufficient). Drought was imposed by withholding watering at 30 days after planting (DAP) - coinciding with flowering. At 20 DAP, growth and N content were significantly higher in NH(4)NO(3)-sufficient plants than in N(2)-fixing and NH(4)NO(3)-limited beans. At later times, only N(2)-fixing and NH(4)NO(3)-sufficient plants continued assimilating N and growing, with the NH(4)NO(3)-sufficient plants being consistently bigger. After 10 days of stress (40 DAP), desiccation was evident, but only NH(4)NO(3)-sufficient plants suffered drought-induced senescence. After 20 days of stress (50 DAP), N content increased in NH(4)NO(3)-sufficient but not in N(2)-fixing beans, despite the latter's lesser state of wilt. Pod dry weight dropped 43% in NH(4)NO(3)-sufficient beans with respect to well-watered plants, while remaining constant in N(2)-fixing beans. Under drought conditions, the number of pods limited pod yield regardless of the nitrogen source used; nevertheless, the translocation of soluble matter to pods continued in both NH(4)NO(3)-sufficient and N(2)-fixing beans. We conclude that common beans grown under conditions of N(2) fixation were more drought tolerant than those provided with sufficient levels of NH(4)NO(3). The most stress-sensitive traits in these plants were the incorporation of N into their shoots and the number of pods remaining on them.

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A collection of rhizobial isolates from nodules of wild beans, Phaseolus vulgaris var. aborigineus, found growing in virgin lands in 17 geographically separate sites in northwest Argentina was characterized on the basis of host range, growth, hybridization to a nifH probe, analysis of genes coding for 16S rRNA (16S rDNA), DNA fingerprinting, and plasmid profiles. Nodules in field-collected wild bean plants were largely dominated by rhizobia carrying the 16S rDNA allele of Rhizobium etli. A similar prevalence of the R. etli allele was observed among rhizobia trapped from nearby soil. Intragroup diversity of wild bean isolates with either R. etli-like or Rhizobium leguminosarum bv. phaseoli-like alleles was generally found across northwest Argentina. The predominance of the R. etli allele suggests that in this center of origin of P. vulgaris the coevolution of Rhizobium spp. and primitive beans has resulted in this preferential symbiotic association.

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A transposon Tn5-induced mutant of Rhizobium meliloti Rm2011, designated Rm6963, showed a rough colony morphology on rich and minimal media and an altered lipopolysaccharide (LPS). Major differences from the wild-type LPS were observed in (i) hexose and 2-keto-3-deoxyoctonate elution profiles of crude phenol extracts chromatographed in Sepharose CL-4B, (ii) silver-stained sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis patterns of crude and purified LPS fractions, and (iii) immunoreactivities otherwise present in purified LPS of the parental strain Rm2011. In addition, Rm6963 lost the ability to grow in Luria-Bertani medium containing the hydrophobic compounds sodium deoxycholate or SDS and showed a decrease in survival in TY medium supplemented with high calcium concentrations. The mutant also had altered symbiotic properties. Rm6963 formed nodules that fixed nitrogen but showed a delayed or even reduced ability to nodulate the primary root of alfalfa without showing changes in the position of nodule distribution profiles along the roots. Furthermore, 2 to 3 weeks after inoculation, plants nodulated by Rm6963 were smaller than control plants inoculated with wild-type bacteria in correlation with a transient decrease in nitrogen fixation. In most experiments, the plants recovered later by expressing a full nitrogen-fixing phenotype and developing an abnormally high number of small nodules in lateral roots after 1 month. Rm6963 was also deficient in the ability to compete for nodulation. In coinoculation experiments with equal bacterial numbers of both mutant and wild-type rhizobia, only the parent was recovered from the uppermost root nodules. A strain ratio of approximately 100 to 1 favoring the mutant was necessary to obtain an equal ratio (1:1) of nodule occupancy. These results show that alterations in Rm6963 which include LPS changes lead to an altered symbiotic phenotype during the association with alfalfa that affects the timing of nodule emergence, the progress of nitrogen fixation, and the strain competitiveness for nodulation.

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Adsorption of Rhizobium meliloti to alfalfa roots before their infection and nodule formation shows the specificity of the symbiotic association (G. Caetano-Anollés and G. Favelukes, Appl. Environ. Microbiol. 52:377-382, 1986). The time course of specific adsorption of R. meliloti (10(3) to 10(4) cells per ml) to roots shows an initial lag period of 3 h, suggesting that either or both symbionts must become conditioned for the adsorption process. Preincubation of R. meliloti L5-30 for 3 h with dialyzed alfalfa root exudate (RE) markedly increased early adsorption of rhizobia to alfalfa roots. The activity in RE was linked to a nondialyzable, thermolabile, trypsin-sensitive factor(s), very different from the root-exuded flavonoid compounds also involved in early Rhizobium-legume interactions. The lack of activity in the RE from plants grown in 5 mM NO3- suggested its negative regulation by the nitrogen nutritional status of the plant. Preincubation of R. meliloti with heterologous clover RE did not stimulate adsorption of rhizobial cells to roots. A short pretreatment of RE with homologous (but not heterologous) strains eliminated the stimulatory activity from solution. The stimulation of adsorption of R. meliloti to alfalfa roots was strongly dependent on the growth phase of the rhizobia, being greater at the late exponential stage. Nevertheless, the capacity of R. meliloti L5-30 to eliminate from solution the stimulatory activity in RE appeared to be constitutive in the rhizobia. The low concentration of rhizobial cells used in these experiments was critical to detect the stimulation of adsorption. The early interaction of spontaneously released alfalfa root macromolecular factor(s) and free-living R. meliloti, which shows the specificity and regulatory properties characteristic of infection and nodulation, would be an initial recognition event in the rhizosphere which triggers the process of symbiotic association.

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Spontaneous mutants of Rhizobium meliloti L5-30 defective in motility or chemotaxis were isolated and compared against the parent with respect to symbiotic competence. Each of the mutants was able to generate normal nodules on the host plant alfalfa (Medicago sativa), but had slightly delayed nodule formation, diminished nodulation in the initially susceptible region of the host root, and relatively low representation in nodules following co-inoculation with equal numbers of the parent. When inoculated in growth pouches with increasing dosages of the parental strain, the number of nodules formed in the initially susceptible region of the root increased sigmoidally, with an optimum concentration of about 10(5) to 10(6) bacteria/plant. The dose-response behavior of the nonmotile and nonchemotactic mutants was similar, but they required 10- to 30-fold higher concentrations of bacteria to generate the same number of nodules. The distribution frequencies of nodules at different positions along the primary root were very similar for the mutants and parent, indicating that reduced nodulation by the mutants in dose-response experiments probably reflects reduced efficiency of nodule initiation rather than developmentally delayed nodule initiation. The number of bacteria that firmly adsorbed to the host root surface during several hours of incubation was 5- to 20-fold greater for the parent than the mutants. The mutants were also somewhat less effective than their parent as competitors in root adsorption assays. It appears that motility and chemotaxis are quantitatively important traits that facilitate the initial contact and adsorption of symbiotic rhizobia to the host root surface, increase the efficiency of nodule initiation, and increase the rate of infection development.

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Bacteria adsorbed in low numbers to alfalfa or clover root surfaces were counted after incubation of seedlings in mineral solution with very dilute inocula (less than 10 bacteria per ml) of an antibiotic-resistant strain under defined conditions. After specified washing, bacteria which remained adsorbed to roots were selectively quantitated by culturing the roots embedded in yeast extract-mannitol-antibiotic agar and counting the microcolonies along the root surface; the range was from about 1 bacterium per root (estimated as the most probable number) to 50 bacteria per cm of root length (by direct counting). This simple procedure can be used with any pair of small-rooted plant and antibiotic-resistant bacterium, requires bacterial concentrations comparable to those frequently found in soils, and yields macroscopic localization and distribution data for adsorbed bacteria over the root surface. The number of adsorbed bacteria was proportional to the size of the inoculum. One of every four Rhizobium meliloti cells adsorbed in very low numbers to alfalfa roots resulted in the formation of a nodule. Overall adsorption of various symbiotic and nonsymbiotic bacterial strains to alfalfa and clover roots did not reflect the specificities of these legumes for their respective microsymbionts, R. meliloti and R. trifolii.

RESUMEN

Early (4 h) adsorption of Rhizobium meliloti L5-30 in low numbers to alfalfa roots in mineral solution was examined for competition with other bacterial strains. All tested competitor strains decreased the adsorption of L5-30 by extents which depended on the strain and its concentration. The decrease of adsorption by R. meliloti competitors (all of them infective in alfalfa) was nearly complete at saturation (97 to 99% decrease). All other heterologous rhizobia and Agrobacterium tumefaciens at saturating concentrations (10 to 10 per ml) decreased adsorption of L5-30 only partially, less than 60%. The differential effects of homologous and heterologous competitors indicate that initial adsorption of R. meliloti to the root surface of its host occurs in symbiont-specific as well as nonspecific modes and suggest the existence of binding sites on roots which are highly selective for the specific microsymbiont in the presence of other heterologous bacteria even in very unfavorable (less than 10) symbiont-competitor concentration ratios.